1. Field of the Invention
The present invention relates to the separation of an animal or plant cell or virus from a biological fluid.
More particularly, the present invention relates to a separating material for use with a cell or virus, a separator for use with the cell or virus, and a method of separating the cell or virus. According to the present invention, the target cell or virus can be separated with high speed, at low cost, providing high and constant recovery or yield and good reproducibility without charging the distribution of target cell subsets. The separating method can be advantageously utilized in various fields, including the medical sciences and biochemistry.
2. Description of Related Art
Recently, in the field of medical science and biochemistry, attempts have been made to separate or isolate a specific group of cells from a suspension containing several different groups of cells, to conduct a basic evaluation of the substances in clinical testing such as immunodiagnosis or immunotherapy. However, there is no method of rapidly separating the target cells at low cost without altering the distribution of target cell subsets, if it is intended to selectively obtain cells such as T, B, K, or NK cells from lymphocytes. As described below, the prior art methods suffer from a number of problems.
Japanese Unexamined Patent Publication (Kokai) Nos. 56-140886 and 57-204454 each disclose the use of a particulate substance containing an acidic functional group or of a hydrophobic and water-insoluble particulate substance with micropores. According to the methods in these Japanese Kokais, it is possible to obtain T cells in a single separation step, but the amount of recovery of the T cells is inconstant and also at a remarkably reduced level. In addition, clogging of the column is caused by the small particle size of the particulate substance used.
Of course, there are some cell separation materials and methods which are internationally recognized to be useful in the area of academic or scientific studies. However, all of these materials and methods required an extended period of time to complete preliminary arrangements for the separation, careful operation of the separation process, and are time-consuming and troublesome. Moreover, they suffer from insufficient reproducibility, because the separation power and separation pattern of the cell separation material used can vary greatly depending on the specific production lot of the material.
Further, Japanese Kokai No. 61-235752 discloses that hydroxyapatite granules have specific adsorptivity to cells. However, their water retention and, accordingly, separation power is rather poor, while a shortened operation time can be obtained. In contrast, Japanese Kokai No. 63-284 states that as a result of using a fibrous apatite, separation power could be improved together with an improved water retention. However, it should be noted that generally a fibrous filler, including the fibrous apatite of Kokai '284, does not result in a uniformly filled state, when packed in a column, and also cannot provide more stable performances in comparison with granular filler, because the performances can vary greatly with changes in the lot used.
More specifically, a "SEPHADEX G10" cell separation method using dextran is well-known in the field of immunodiagnosis. Although the principles of this method have not been completely clarified, it is considered that the level, i.e., higher or lower, of the adhesion properties of the cells play a leading role in this method. Namely, macrophages or adhesive accessory cells having a large size are separated by adsorption, while T cells and B cells pass through the column. However, it is essential to this method that small non-adsorptive accessory cells pass through the column, and some of the subsets of the T cells adhere to the filler. Because of the undesirable adhesion of the T cells, it is impossible to obtain a complete population of the T cells. These problems must be solved to effectively carry SEPHADEX G10 method in the immunodiagnosis.
Furthermore, use of a separation column containing a filler of nylon wool is also well-known. This column is generally used to separately obtain T cell-rich cell groups, but the effective recovery or yield of the target T cells is relatively low, in the range of 12 to 25%. Also, while the T cells can be obtained at a relatively high purity, the distribution of the T cell subsets is changed after passing through the column. Namely, the distribution of the separated T cell subsets is not the same as that of the T cell subsets in the starting cell suspension. In addition to this variation of the cell population, the separating power and separation pattern may also vary depending upon various factors such as the specific lot of nylon wool selected, the specific splitting method of the wool, the specific filling method, and the specific washing method, even though the wool is filled in the column after the weighing.
As is appreciated from the above discussion, at present, the cell separation technologies based on the adsorptive properties of the cells are merely at a starting point in terms of practical use. It is therefore desired to improve the cell separation materials and methods and to develop a novel separation material, thereby ensuring high efficiency, speed, and accuracy in separation of the cells. If this improvement and development is attained, the cell separation technologies will be more widely and more advantageously utilized in various important fields such as biotechnology and medical science, for example, in immunodiagnosis and immunotherapy.
In addition to cell separation, an improvement in virus separation is also an important goal in the fields of medical science and biochemistry. Since the virus separation is essential to prophylaxis and treatment of viral diseases including influenza, and recently carriers of acquired immune deficiency syndrome (AIDS) virus are rapidly increasing, improvements in virus separation are particularly of urgent necessity for medical care. At present, particularly with regard to the separation of the hepatitis B virus, several virus separation technologies using a polymeric membrane, hollow fiber, or ion exchange resin are well-known. However, these separation technologies entail complicated operation and expensive separation devices due to the specific separation systems thereof. This is the reason why these technologies have not yet been put to practical use.
As previously discussed, Japanese Kokais Nos. 61-235752 and 63-284 teach the use of hydroxyapatite granules in cell separation, but suffer many drawbacks and do not suggest that their teachings may be applied in virus separation. Moreover, Japanese Unexamined Patent Publication (Kokai) No. 63-16045 discloses porous granules of calcium phosphate as a filler in liquid chromatography. The calcium phosphate granules can be used in the chromatographic column to selectively separate and purify a biopolymer such as a protein. However, this Kokai is silent on the subject of use as an adsorption or separation agent for cells or viruses.